Investigation of autophagy as a survival factor for chronic myeloid leukaemia

Tyrosine kinase inhibitors (TKIs) have revolutionised the treatment of chronic myeloid leukemia (CML), however, fail to cure the disease due to the persistence of a refractory fraction of stem/progenitor cells. Autophagy is a recycling mechanism utilised by the cell as a survival mechanism under stressful conditions, and its induction has been suggested to have a cytoprotective role in cancer cells. In this study we demonstrate that autophagy is triggered in CML upon TKI-mediated inhibition of BCR-ABL, and protects from cell death. In order to evaluate if specific autophagy inhibition enhances TKI effects, we stably transduced primary CML stem/progenitor cells with a vector carrying a short-hairpin against the key autophagy gene ATG7. Knock-down of basal ATG7/autophagy levels in CML stem/progenitor cells inhibited by approximately 50% the survival of the cells in a clonogenic assay, and reduced by 75% their erythroid differentiation potential. Furthermore, ATG7 knock-down enhanced the effects of TKIs imatinib (IM; 1st), dasatinib (DAS; 2nd), nilotinib (NIL; 2nd) and ponatinib (PON; 3rd generation), reducing by 92-98% the survival of these cells in a clonogenic assay. In contrast, ATG7 knock-down in normal stem cells, with or without TKI treatment, did not have a significant effect on survival and proliferation. ATG7 was also knocked-down in final disease stage, blast crisis (BC), patient-derived K562 and KCL22 cell lines. Both cell lines appeared to depend significantly on autophagy for survival as indicated by high apoptosis levels (70-100%) after ATG7 knock-down. Interestingly, ATG7 knock-down cells appeared to be more differentiated compared to the control (scrambled shRNA). Our findings suggest a role for basal autophagy in the survival, differentiation decisions and clonogenicity of CML cells, and support the combined use of autophagy inhibition with TKIs for the eradication of CML stem/progenitor cells. This could be partially attributed to a bypass of the differentiation block upon autophagy inhibition, which facilitates TKI-targeting. We underline the necessity for the development of specific autophagy inhibitors that in combination with TKIs could potentially eradicate the fraction of persistent CML stem/progenitor cells and offer a curative option for CML patients

Μεσογειακή Διατροφή και Χημεία: Διερεύνηση των απόψεων και αλλαγών στην εκπαίδευση και τις διατροφικές συνήθειες μαθητών λυκείου την περίοδο της πανδημίας Covid-19

Πρόσφατες επιστημονικές μελέτες έχουν αναδείξει τον ρόλο της Μεσογειακής Διατροφής (ΜΔ) στην πρόληψη των επιβλαβών επιπλοκών της νόσου COVID-19 μέσω των αναστολέων του φλεγμονώδη παράγοντα PAF (Platelet-Activating Factor), που εμπεριέχονται στα μικροθρεπτικά συστατικά της. Επιπλέον, η πανδημία COVID-19 άλλαξε σημαντικά τις καθημερινές συνήθειες των εφήβων μέσω της απουσίας δια ζώσης διδασκαλίας και σχολικών δραστηριοτήτων λόγω του παρατεταμένου απαγορευτικού (lockdown). Δεδομένου ότι στη βιβλιογραφία δεν υπάρχουν αρκετά στοιχεία για την Ελλάδα, στόχος της έρευνας ήταν να αποκαλύψει τις αλλαγές στη διατροφική κατάσταση και την καθημερινότητα των μαθητών λυκείου πριν την πανδημία και κατά την διάρκεια του lockdown, και να αξιολογήσει τις διατροφικές τους συνήθειες μέσω της κλίμακας προσκόλλησης στη ΜΔ ‘KIDMED’. Η έρευνα βασίστηκε σε ερωτηματολόγιο το οποίο διαμοιράστηκε σε 207 μαθητές λυκείου τον Μάϊο του 2021 (αμέσως μετά τη λήξη του 6μηνου lockdown). Τα σημαντικότερα αποτελέσματα της μελέτης συνοψίζονται ως εξής: (i) 42% της Γ’ Λυκείου δείχνει χαμηλό ενδιαφέρον για το μάθημα της Χημείας , (ii) >50% των μαθητών θεωρεί ότι το lockdown είχε αρνητική επίδραση στην διατροφή, (iii) πριν την πανδημία ο τροποποιημένος δείκτης KIDMED ήταν κοντά στο άνω όριο της Β’ Βαθμίδας (4,72), ενώ κατά την περίοδο του lockdown μειώθηκε σημαντικά προς το κάτω όριο (3,18), (iv) βρέθηκε στατιστικά σημαντική συσχέτιση μεταξύ του δείκτη KIDMED και της ενημέρωσης για τα οφέλη της ΜΔ καθώς και της συμμετοχής σε αθλητικές δραστηριότητες (πριν και μετά), (v) πριν το lockdown ο δείκτης KIDMED ήταν στατιστικά μεγαλύτερος για τους μαθητές που δεν αφιέρωναν χρόνο για ψυχαγωγία με ΗΥ, και (vi) κατά την περίοδο του lockdown ο δείκτης KIDMED της Α’ Λυκείου ήταν στατιστικά μικρότερος σε σχέση με την Β’ & Γ’ Λυκείου (2,72 vs. ≈3,5). Συμπερασματικά, η πανδημία επηρέασε αρνητικά τις διατροφικές συνήθειες των μαθητών και των τριών τάξεων του λυκείου ενώ οι μαθητές που είχαν ενημερωθεί για τα οφέλη της ΜΔ κατά της νόσου COVID-19 είχαν αυξημένο δείκτη KIDMED. Εκπαιδευτικά προγράμματα που προάγουν το μεσογειακό πρότυπο διατροφής σε συνεργασία με το οικογενειακό περιβάλλον θα μπορούσαν να προσφέρουν σημαντικά στην υιοθέτηση ενός υγιεινού τρόπου ζωής.Recent scientific studies have highlighted the role of MD in preventing the complications of COVID-19 via the inhibitors of the inflammatory factor PAF (Platelet-Activating Factor), which are contained in the MD micronutrients. In addition, the COVID-19 pandemic significantly altered the adolescents' daily habits due to the lack of face-to-face teaching and school activities as a result of the prolonged lockdown. Given that the scientific literature lacks information about the effect of COVID-19 pandemic in Greek adolescents, the aim of this research was to reveal potential changes in the nutritional status and daily life style of high school students before the pandemic and during the lockdown, and to evaluate their nutritional habits using the Mediterranean Diet Quality Index 'KIDMED'. The research was based on a questionnaire that was distributed to 207 high school students in May 2021 (immediately after the end of the 6-month lockdown). The most important results of the study can be summarized as follows: (i) 42% of the 3rd Grade students shows low interest in the course of Chemistry, (ii) >50% of the students considered the lockdown to have negative effect on their diet, (iii) before COVID-19 the modified KIDMED score was close to the upper limit of Level 2 (4.72), while during the lockdown period it decreased significantly to the lower limit (3.18), (iv) a statistically significant correlation was found between the KIDMED score and information about the benefits of MD, and participation in outdoor activities (both before and after the lockdown), (v) before the lockdown the KIDMED score was statistically higher for students who did not spend time on PC entertainment, (vi) during the lockdown period the KIDMED score of Grade A students was statistically lower than Grade B & C students (2.72 vs. ≈3.5). In conclusion, the present study highlights the negative effect of the lockdown in the dietary habits of high school students, whereas students who were informed about the benefits of MD showed an increased KIDMED score. Educational programs that promote the Mediterranean diet in collaboration with the family environment could make a significant contribution to the adoption of a healthy lifestyle

Hydroxychloroquine for chronic myeloid leukemia: complete cure on the horizon?

No abstract available

Widespread mitochondrial depletion via mitophagy does not compromise necroptosis

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death

Response surface optimisation for the extraction of phenolics and flavonoids from a pink guava puree industrial by-product

Pink guava puree industry produces huge amount of by-products that have potential as sources for polyphenols. Response surface methodology was implemented to optimise the extraction conditions for phenolics (Y1) and flavonoids (Y2) from a by-product of the guava industry. A three-factor inscribed central composite design was employed to determine the effects of three independent variables, namely pH (X1: 2-6), temperature (X2: 40-60 °C) and time (X3: 1-5 h), on the response variables. The corresponding predicted values for phenolics and flavonoids were 336.30 and 427.35 mg 100 g-1, respectively. Predicted values for extraction rates of phenolics agreed well with experiment values; R2 of 0.902. However, the model derived for flavonoids extraction was less reliable; R2 of 0.983. Increase in time and temperature was found significant in increasing the extraction rate. The optimum conditions for extracting phenolics by ethanolic solvent occurred at a pH of 2 and 60 °C for a 5-h extraction

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence

Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing

Background As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients’ samples. Method We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals’ sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. Results Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0–60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6–56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13–60 vs. pooled sputum MAR 25.3, SD 14.6, range 1–54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12–3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. Conclusion We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population “bubble”

Understanding the ellagitannin extraction process from oak wood

[EN] The extractability of the main oak ellagitannins has been studied in five model solutions containing different types of oak chips (two sizes and different toasting degrees for each size). A new extraction kinetic model has been proposed from the quantitative experimental results obtained by means of HPLCeESI-MS/MS-multiple reaction monitoring method. The model considers an initial extraction (i.e., washing step) followed by a diffusion step, which involves two different processes that follow first-order kinetics at different rates. Differences in the extractability of the ellagitannins in the different model solutions have been observed and explained on the basis of the kinetic model here proposed

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia chromosome-positive cells

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease